Heparin is widely used as an anticoagulant. Heparin is used in an injectable form to prevent or treat blood coagulation in patients suffering from, or at risk of suffering from, blood clots. Heparin is also used to form an anticoagulant surface on various experimental and medical devices such as, for example, renal dialysis machines.
Heparin is a polysaccharide which is one of four main subgroups of glycosaminoglycans (GAGs). GAGs are long polysaccharides that are made up of repeating disaccharide units. Within the GAG family there are four main subgroups: (1) hyaluronic acid or hyaluronan (HA); (2) keratan sulfate (KS); (3) chondroitin/dermatan sulfate (CS/DS); and 4) heparan sulfate (HS)/heparin. These subfamilies differ in monosaccharide components, glycosyl linkages, and position and degree of saccharide functionalization. With the exception of HA, GAGs are found covalently attached to a protein core and together are known as proteoglycans.
HS and heparin comprise the disaccharide units α(1→4) linked D-glucosamine (GlcN) and (1→4) linked uronic acid which is either α-linkage L-iduronic acid (IdoA) or β-linkage D-glucuronic acid (GlcA). Heparin and HS are heterogeneous in nature not only due to differing disaccharide combinations but also their sulfate modifications. Possible modifications include 2-O-sulfation of uronic acid, and N-sulfation, N-acetylation, 3-O- and 6-O-sulfation of glucosamine.
Almost every mammalian cell produces GAGs which are incorporated into proteoglycans in a cell-associated glycocalyx that populates the extracellular matrix (ECM) to define tissue form and function. HS has been shown to modulate cell growth and development by regulating growth factors such as the fibroblast growth factor (FGF) family, platelet derived growth factor (PDGF), and vascular endothelial cell growth factor (VEGF). Heparin, however, is produced predominantly in mast cells.
GAG synthesis requires the precise and timed activity of many enzymes that are involved in GAG chain elongation, epimerisation of the glucuronic acid moiety and modification by sulfates at distinct sites in the chain, a process that is not template-driven and thus results in chain heterogeneity.
The initial product in the biosynthesis of HS and heparin is polysaccharide composed of alternating glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) residues which is covalently bonded to a protein core via a linkage tetrasaccharide covalently bonded to the protein via an oxygen moiety at a serine residue. The serine residue is part of a GAG attachment sequence. The attached polysaccharide is then modified by enzymes in the cell to form complex sulfated derivatives.
The first step in the synthesis of the HS and heparin chain is the formation of a linkage tetrasaccharide which is initiated by the coupling of a xylose to the serine residue of an attachment sequence via the action of xylosyltransferase (XylT), followed by the addition of two galactose molecules in sequence by galactosyltransferases (GalT) 1 and 2, respectively. The tetrasaccharide structure, which is also common to CS chains, is completed by the addition of GlcA by glucuronic acid transferase (GlcAT) 1. It is at this point in GAG biosynthesis that the HS/heparin biosynthetic pathway diverges from the CS/dermatan sulfate (DS) pathway by the addition of an N-acetylglucosamine (GlcNAc) instead of a N-acetyl galactosamine (GalNAc), though the exact mechanisms responsible in controlling this switch between the major GAG types is unknown.
The enzymes that are responsible for the addition of the GlcNAc to the non-reducing end of the linkage tetrasaccharide are known as the EXTL family of glycosyltransferases. The polymerization or elongation of the HS/heparin chains involves a complex being formed between EXT1 and EXT2 that sequentially add a GlcA followed by a GlcNAc moiety and this gives rise to the characteristic alternating copolymer of these two monosaccharides seen in all HS and heparin chains. The sulfation of HS/heparin is started when the acetyl group on some GlcNAc residues is removed and sulfated by one of the four isoforms of the N-deactylase N-sulfotransferase (NDST) enzymes. NDST2 has been shown to have specificity for modifying the GAG chains decorating serglycin making it a critical enzyme in the process of synthesizing mast cell heparin. After this sulfation step, further modification of the HS/heparin chains happens in close proximity to these regions leading to relatively highly sulfated domains or S domains. Most forms of HS contain approximately 30% of their sequence decorated with sulfate leaving approximately 70% as undecorated GlcA-GlcNAc sequences whereas heparin contains long stretches of highly sulfated disaccharides. Once the N-sulfate modification has taken place, some of the GlcA residues in these regions become epimerized to iduronic acid (IdoA) followed by the vast majority being sulfated at the C2 position. The glucuronyl C5-epimerase and hexuronyl 2 sulfotransferase enzymes both have only a single isomer and co-localize in the Golgi, and they have been shown to interact with each other as well as the interaction between C5-epimerase and 6-O-sulfotransferase. Sulfate groups can be added to the C6 position of either GlcNAc or GlcNS by one of three isoforms of the 6-O-sulfotransferase enzymes. These enzymes can act on both GlcNAc and GlcNS but have been shown to have a preference for modifying regions where there is a higher proportion of GlcNS flanked by 2 sulfate modification residues, which supports the synthesis of longer S domains.
The complexity of GAG synthesis, and the lack of information in relation to the conditions which influence attachment of GAG chains to the protein core, presents a challenge for the production of proteoglycans with heparin or HS chains.
Pharmaceutical-grade heparin is still derived from animal tissues such as porcine (pig) intestines or bovine (cattle) lungs. However, heparin obtained from these sources has been associated with some adverse events due to doping with oversulfated chondroitin sulfate (Guerrini et al. Nat. Biotechnol. 2008, 26, 669-675). In addition, isolation of heparin from animal tissue runs the risk of transmission from animals to humans of disease agents such as, for example, viruses, bacteria and TSE. Alternative methods of producing heparin such as chemical or chemo-enzymatic synthesis can produce well-defined structures but are yet to produce the heterogeneous population of structures present in animal-derived heparin.
What are needed are alternative methods for production of heparin and HS. The present disclosure provide novel methods for the production of heparin and/or HS that address the shortcomings of the prior art.